Complex II is one of the four electron transfer enzymes that generate the transmembrane electrochemical gradient in mitochondrial aerobic respiration. Its genetic alteration or inhibition has a significant effect on survival of the organism. The complex II superfamily is perhaps the most versatile respiratory enzyme since it is involved in aerobic and anaerobic respiration and the Krebs cycle. The recycling of the complex II architecture for multiple purposes suggests that this fold is evolutionary ancient. Examination of this single enzyme can give insight into biological mechanisms of fumarate reduction, succinate oxidation, aspartate oxidation, quinol chemistry, electron transfer, and the formation of the transmembrane electrochemical gradient. In preliminary studies, the complex II homolog quinol:fumarate reductase has been overproduced, crystallized and the structure determined alone and in complex with inhibitors of the quinol-binding site. Further, the structures of several mutagenic variants have been determined, revealing unexpected conformational rearrangements that give insight into enzyme activity. These preliminary results allowed us to generate a hypothesis for the catalytic reaction mechanism and that includes accompanying conformational motion. This sets the stage for the Aims of this proposal: In Aim 1, we will describe the chemical details of catalysis at the dicarboxylate-binding site. To achieve this, we will co-crystallize the quinol:fumarate reductase (QFR) with multiple small molecules and structurally investigate mutants that alter the catalytic efficiency. In Aim 2, we will characterize the motions associated with catalysis. We have already classified enzyme motions into three distinct types - interdomain motions, motions of a catalytic loop, and global motions. We have begun structural characterization of a site-directed mutant that interrupts normal domain motion. Complemented by computational methods that evaluate the energies of each trapped state, we will further characterize the large, interdomain motion using a combination of site-directed spin labeling and electron paramagnetic resonance spectroscopy. In Aim 3, we will investigate the influence of environmental fungicides on the catalytic activity of complex II. Here, we will co-crystallize several quinol-binding inhibitors with both the wild type E. coli QFR and a variant of the E. coli QFR that has fungicide binding properties similar to the human enzyme. We will further work to express human complex II with a heterologous system for structural studies.